Tick-borne diseases, including anaplasmosis, ehrlichiosis, and several rickettsial diseases, remain poorly controlled amid increasing incidence. The interruption of tick transmission is a potential critical control point for disease prevention. However, the mechanisms by which these pathogens are able to successfully colonize the tick represent a significant gap in our knowledge, impeding the ability to exploit this control point in disease prevention. The goal of the proposed research is to identify pathogen molecules required for colonization of and successful transmission by ixodid ticks. Pathogen transmission relies on the successful transition from the vertebrate host into the tick, which includes the entry into and replication within radically different host cell types including cells of the tick midgut and salivary glands. Through transcriptomics and proteomics, pathogen proteins specifically up-regulated during tick colonization have been identified for both Anaplasma and Ehrlichia. However, the genetic tools needed to test the functional requirement of individual proteins and functional pathways are either unavailable or in development for these organisms. In contrast to this limitation for tick-borne rickettsial pathogens, exploitation of genomic sequence data through mutant library screens has allowed for rapid identification of genes required for specific functions in a broad array of bacterial pathogens. Using a transposon mutant library of Francisella tularensis subsp. novicida, we established that F. novicida can colonize Dermacentor andersoni nymphs, and survive within the tick midgut during the molt to adulthood. Once the adult ticks are fed on a second host, the F. novicida disseminate to the tick salivary glands, and are transmitted to the mammalian host, thus completing a transmission cycle similar to Anaplasma, Ehrlichia, and Rickettsia. Importantly, colonization of the tick by mutants is non-random; thus, unless a defect is present which specifically affects the ability to colonize the tick, the population of mutants colonizing the tick will reflect the population of mutants in the blood meal. This allows for the specific detection of mutants unable to colonize and thus genes required for transmission. We hypothesize that the required genes will group into functional pathways broadly applicable to intracellular, tick-transmitted pathogens. Through both targeted gene knockout and cross-species complementation experiments, broadly conserved requirements for tick transmission can be identified. This research is consistent with the exploratory/developmental nature of the R21 mechanism because it utilizes a new model system to search for genes and gene pathways required for transmission. The proposed studies may lead to novel and potentially broadly applicable strategies to prevent the transmission of tick-borne diseases. PUBLIC HEALTH RELEVANCE: According to the most recent data available from the CDC, the incidence of tick-borne disease, including anaplasmosis, ehrlichiosis and Rocky Mountain spotted fever, has been steadily increasing (CDC tick diseases website). The objective of this research is to identify how bacterial pathogens colonize the tick, with the long-term goal of developing new means of preventing tick colonization, and thus preventing tick-borne disease.